PT96503B - PROCESS OF PREPARATION OF CONJUGATES OF POLYMEROS AND POLYNUCLEOTIDOS BIOLOGICAMENTE ESTAVEIS FOR THE TREATMENT OF SYSTEMIC ERITEMATOSOUS LUPUS - Google Patents

Abstract

Chemically defined conjugates of biologically stable polymers, such as copolymers of D-glutamic acid and D-lysine, and polynucleotide duplexes of at least 20 base pairs that have significant binding activity for human lupus anti-dsDNA autoantibodies. The duplexes are preferably homogeneous in length and structure and are bound to the polymer via reaction between an amino-reactive functional group located at or proximate a terminus of each duplex. These conjugates are tolerogens for human systemic lugus erythematosus.

Description

description

Technical Field

This invention relates to compositions for the treatment of systemic lupus erythematosus, autoimmune disease (SLE or lupus). More particularly, it relates to conjugates of biologically stable polymers, preferably copolymers of D-glutamic acid (represented here by the simple designation letter E) and D-lysine (represented here by the simple designation r letter and certain polynucleotides that have been found to be effective for inducing tolerance for antigens involved in SLE The preferred copolymers are represented here by the designation D-EK.

Background

Immunotolerance describes the mechanism that leads to the often permanent and long-lived form of immunosuppression that prevents individuals from reacting with their own tissues. It is believed that immunotolerance to the antigens themselves (auto-antigens) is usually established during neonatal development and persists throughout the animal's life. Not surprisingly, however, this scheme is sometimes imperfect and some individuals, typically late in life, acquire autoimmune diseases. One such disease is SLE. It is characterized by the production of autoantibodies to the individual's DNA, and results in progressive inflammatory degeneration of the kidneys.

SLE is typically treated by administering a broad spectrum of non-specific immunosuppressants, such as cyclophosphamide or prednisone. Because these dredgers often suppress all aspects of the immune system, they suppress their necessary and beneficial functions, as well as the poor function that causes SLE. Therefore, they must be administered with extreme caution and are not always appropriate to treat the disease in a continuous manner. In addition, individuals who are generally and often immunosuppressed by drug treatment are at risk for other complications, especially infectious diseases. s

A preferable attempt for the treatment of SLE would be to administer the drug that is capable of restoring immunotolerance to the antigens involved in the SLE without affecting the normal functions of the immunosystem. Unfortunately, there is no common way of treating SLE, or any type of autoimmune disease, that is favorable and specific for the autoantigens associated with the disease. The conjugates of the invention are a means to • provide such treatment for SLE »

Benacerraf, Katz and their colleagues investigated and described the use of D-EK conjugates with haptens and various antigens to induce specific immunotolerance. Their initial studies involved 2ji4-dinitrophenyl synthetic hapten (DNP) conjugates in guinea pigs and rats, and showed that the conjugates could induce tolerance to

DNP. These initial studies were extended to other haptens / antigens such as tasna E and benzylpenicilloid (BF'O) antigens. See U.S. Patents No. 4,191,668 and

4,220 «565 u

U.S. Patent 4,191,668 (Example IV) describes the preparation of D - EK conjugates and oligonucleotides isolated from DNAse purified DNAse thymus DNA. Oligonucleotides were characterized as being made up of less than 10 nucleotides. Although column 11 of US Patent 4,191,668 indicates that their invention has therapeutic values for the treatment of autoimmune disease and mentions SLE, data on the immunological effects of oligonuc 1 and D-conjugates have not been provided. -EK men ci on ad os „

Katz and his colleagues also investigated the potential of nucleoside-D-EK conjugates to induce tolerance to the determinant nucleic acids. Eshar et al. In J. Immunology (1975) 1145 872 - · 876. In this matter in question, individual nucleosides are widely believed to be the main determinants of specificity in lupus antisera. They administered conjugates of the D-EK copolymer and four ribonucleosides to the SJL or Balb / c rat,

subsequently immunized the rats, treated with a lancet lancet hemocyanine (KI-IL) -ribonucleoside conjugates. In both species, sera-antinucleoside antigen-binding capacity has been decreased to simply detectable levels. While these studies showed that such conjugates could produce nucleotide immunotolerance, they did not show that such conjugates were effective in treating SLE.

Other researchers have studied nucleoside or DNA conjugates with other transporters »Borel et al. (Science (1973) 182 to 76) evaluated the ability of isogenic mouse IgG-nucleoside conjugates to reduce the antibody response to denatured DNA in young animals of the NZB rat species. This species is used as a model for some autoimmune phenomena. They tend to produce antibodies to key nucleic acids that form immune complexes that lodge in the kidneys and lead to glomerular nephritis. In these studies, treated animals produced significantly reduced levels of anti-denatured DMA antibodies and exhibited less membranous glomerular nephritis. than control and free animals treated with nucleosides. In separate studies, Parker et al. (J. Immunol (1974) 113; 292) evaluated the effects of denatured DNA conjugate for pol i - DI i si on and / or cyclophosphamide on the progression of the syndrome described above in NZB rats . These studies demonstrated a significant increase in survival and a significant decrease in the DNA binding capacity for treated animals when compared to controls. None of these studies, however, were aimed at producing

tolerance to .dsDNA which appear to be the major antigens involved in human SLE.

In a later article (Ann Ny Acad Sc.i. (1986) 475; 296306) Borel et al. Suggest that the performance of SLE-specific immunotherapy has been hindered by an inability to bind DNA fragments to soluble proteins. Citing the previous works by Stoller (Papalian et al; J. Clin Invest (1980) 65 s 469 and Stoller and Papalian, J »Clin Invest (1980) 66 s 2: 1.0), the authors state that a minimum size of at least At least 10 - 40 base pairs of DNA is required to bind the anti-DNA antibody produced in SLE patients. The article describes immunoglobulin-aligonucleotide conjugates made by binding a fraction of native DMA slightly larger than 10 base pairs using glutaraldehyde as a binding agent. The Figure in the article describes the studies used to select the DNA fraction. That Figure describes the reactivity of the various DNA fractions linked to the red blood cells of the sheep, via g-1 utaralcid with anti-DNA antibodies in the sera BWF. In these 1 tests, the fraction designated 70 - 80 was the most reactive. 0) such a fraction is described as being slightly larger than the fraction 81 - 101 which corresponds to about 10 oligonucleotides. The next largest fraction with respect to 70 80, designated 40 - 69, exhibited significantly reduced reactivity, with respect to the fraction 70 - 80 »It will be appreciated that the fraction slightly larger than 10 base pairs is heterogeneous in such size and due to the binding, is linked to immunoglobulin at a random location in the chain. In addition, because the bifunctional binding agent is used, it is likely that some ο

degrees of cross-1inking have occurred in the coupling reaction. Thus, the conjugate described in this article is not a chemically defined half insofar as (a) the length of the oligonucleotide is not specified, (b) a fraction of the oligonucleotide contains chains of variable length, (c) the site of binding to the immunoglobulin, along the length of the oligonucleotide chain is random (d) there are a few degrees of cross ..... linking, and (e) non-oligonucleotides conjugated but cross-linked may not be separated from the conjugated material.

Borel et al. Have recently (J. Clin Invest <1988 §2 = 1901 - 1907) reported in vitro studies using linked human immunoglobulin conjugates, either the total synthesized DMA (designated N), or 20 - 30 fractions base pairs

10-100 (designated N) using glutaraldehyde as a binding agent.

20-30

The conjugates have been reported to exhibit genetically tolerable properties in vitro in PBL’s of SLE patients.

These conjugates, however, similar to that reported in his 1986 article, are also produced with heterogeneous mixtures of oligonucleotides using methods that produce, not specifically, cross-1inking chains. From this point on, neither the chemistry nor the biological activity of these conjugates would be sufficiently reproducible to allow them to be approved as pharmacists.

Discovery of the invention

In contrast to the prior art, applicants have. developed chemically defined conjugates of biologically stable polymers, and polynucleotide duplexes that are genetically tolerable for human SI..E. These duplexes are defined with respect to length, polymer binding site, helical structure, mandatory affinity for human SLE anti-dsDNA antibodies. Consequently, their chemical and genetic tolerance activities are reproducible to a degree that makes these conjugates responsible for quality control and approval as pharmacists.

Thus, one aspect of the invention is a conjugate of a biologically stable polymer and a multiplicity of polynucleotide duplexes of at least about 20 base pairs each linked to the polymer, significant binding activity for the anti-dsDNA autoantibodies of the Human SLE. In a preferred configuration of these conjugates, the duplexes are substantially homogeneous in length and are coupled to the polymer at or near (i.e., within about 5 base pairs) to one of its ends, such that each duplex forms a pendant chain , of at least about 30 base pairs, measured from the place of binding of the duplex to the polymer until the free end of the chain.

Pharmaceutical compositions containing these conjugates and methods for treating SLE using these conjugates are other aspects of the present invention.

Yet another aspect is a conjugate of (a) a biologically stable polymer and (b) a multiplicity of polynucleotide duplexes, each of which is all linked to the polymer by a functional group located at or near the terminus of one of the duplex chains, said conjugate being genetically tolerable to human SLE.

A further aspect of the invention is a method for making the conjugates described above, comprising: reacting a multiplicity of single-stranded polynucleotides, each of which is at least about 20 nucleotides in length and has a functional group in, or near one of its terminus, which reacts with free amino groups in the polymer, to form a conjugate and complementarily pair single-stranded polynucleotides to single-stranded polynucleotides, conjugated to the polymer to form pendant double-stranded DNA strands.

J

Brief Description of the Figures

Figure 1 is a graph of the data obtained through the tests described in Example 1.

Figures 2 and 3 are reproductions of the CD spectrum described in Example 3.

Figure 4 is a graph comparing the SLE antisera's binding capabilities to DNAs having different types of helical configurations.

Figures 5 - 8 are graphs of data obtained in the J tests described in Example 5.

Modes of Carrying Out the Invention □ the polymer component of the conjugate is biologically stable, that is, it exhibits in vitro an excretion half-life of days to months. These polymers are also substantially non-immunogenic (ie, they exhibit no or only weak immunogenicity when administered to animals) and are

preferably consisting of a simple synthetic chain of defined composition. They will normally have an average molecular weight in the range of about 5,000 to about 200,000, preferably 5,000 to 50,000. Examples of such polymers are palietylene glycol, polyisin, polyvinyl alcohol, polyvinylpyrrolidone, immunoglobulins, and D-EK. Particularly preferred polymers are D-EK's having a molecular weight of about 5,000 to about 50,000 and an E: K molar ratio of approximately 60:40.

Synthetic polynucleotide duplexes, which are coupled to biologically stable polymers, are composed of at least about 20 bp, more usually at least 30 bp, typically 30 - 250 bp, and preferably 50 -150 bp

Preferably, the duplexes are substantially homogeneous in length; that is, the variation in length in the population will not normally exceed about ± 207., preferably ± 107, of the average length of the base pair duplex. They are also, preferably, substantially homogeneous in the nucleotide composition; that is, their base composition will not vary by more than about 107. More preferably, they are entirely homogeneous in their nucleotide composition. In terms of composition, the preferred synthetic or recombinant dsDNA is preferably made up of chains of repeating mer units, complementary to 2 - 4 bases (i.e., a repeating dimer, trimers or tetraimers), such as (AC) (dimer) (TG) (TAC) (trimester)

(ATG) (GCTA) (tetraomer) n '' (CGAT) n '?

where n, n ', en ^: ' · are whole numbers selected to provide the desired number of base pairs. Polynucleotides made up of isomeric dimers, for example, poly d (AC) s poly d (GT) and poly d (AG) s poly d (CT) are most preferred.

Based on the interpretation of the circular dichroic spectrum (CD), it is believed that the duplexes, which are useful in the invention, assume a typical B-DNA helical structure. It should be understood that it is not intended that the invention be limited by these convictions and that the duplexes can, after more conclusive analysis, assume helical structures typical of Z-DNA and / or A - DNA. The sense helices of the B-DMA-shapes, having base pairs approximately perpendicular to the longitudinal helical axis of the other two types of DNA helices. The helical structures of the different types of DMA can be characterized by the circular dichroic spectrum (CD). The CD spectrum of B forms of DMA exhibits (: l.) Positive dichroic bands associated with sense helicality in portions of the spectrum below 250 nm, but separated from dichroic bands of positive long wavelength greater than 206 nm by a significant minimum at a wavelength between 240 and 260 nm, and (2) a simple sharp peak, greater than 250 nm, with a maximum blue jump relative to the maximum seen in the spectrum of forms A of the

RNA, and DMA and centered at a wavelength between 270 and 290 nm. Through a general comparison of the two helical forms of DNA, Ζ-DNA is distinguished by its curve r

reverse helical helix with unpositioned base pairs, symmetrically around the helix, and the A ~ DNA forms a more open helix in the forward direction, in which the base pairs are oriented abliquely to the longitudinal helix and are pulled towards outside the center of the propeller.

These polynucleotide duplexes can be synthesized from native DNA, or synthesized by chemical or recombinant techniques. The dsDNA, naturally occurring or produced recombinantly, of long length, can be purified (for example, enzymatically, chemically, or by mechanical fission) and fractionated (for example, by agarose gel or Sephadex column), to obtain polynucleotides of the length wanted.

Alternatively, single and complementary polynucleotide strand pairs, chained up to about 70 bases in length, are prepared quickly using commercially available DNA synthesizers, and then paired to form duplexes by conventional procedures. Synthetic dsDNA of longer length may be obtained by enzymatic extension ^(5! 'Ization one phosphinyl ori binding hereinafter) of small strands produced chemically.

Polynucleotides can also be produced by molecular cloning. For example, oligonucleotides of desired length and sequence are synthesized as above. These oligonucleotides can be designed to have appropriate termini for binding at specific restriction sites. Multiple repetitions of these oligomers can be linked in a row to promote replication of multiple copies. The resulting constructs are inserted into a standard cloning vector, and the vector f is introduced into a suitable cell / microorganism by transformation. The transformed ones are identified by standard markers and are increased under conditions that favor DNA replication. Polynucleotides can be isolated from the other DNA of the cells / microorganisms by treatment with restriction enzymes and fractionation of conventional size], (for example, agarose gel, Sephadex columns).

Alternatively, oligonucleotides can be replicated by polymerase chain reaction (f'CR) technology. Saiki, R »K., et al., Science (1980) 230; 1550; Sacki, et al., Science (1988) 259a487; Sambrook, et al., In Molecular Cloning Techniques: A Laboratory Manual, vol. 12, p. 14, 1 - 14, 35 Cold Spring Harbor Press (1989),

In contrast to the prior art conjugates, each polynucleotide duplex employed in the present invention exhibits significant binding activity with the antisera SLE.

Preferably, they are substantially homogeneous in length. In this respect, polynucleotides of the prior art were heterogeneous in length and comprised of a mixture of chains, some or all of which are too small to exhibit such activity. Polynucleotides can be screened for binding activity with the antisera SLE by the assays described in the examples. The modified Farr assay, in which binding activity can be expressed as I (the polynucleotide concentration, in molar nucleotides, resulting in half the maximum inhibition) was a preferred assay. Polynucleotide duplexes have an I of less than about 500nM, preferably less than 50nM, are considered to have significant binding activity and are therefore useful for the manufacture of the conjugates of this invention.

Polynucleotides are attached to the polymer in a way that preserves its binding activity. This was done by coupling the polynucleotide to the polymer at a predetermined location on the polynucleotide chain, such that the polynucleotide forms a pendant chain, of at least about • 30 base pairs measured from the coupling site to the free end (not attached) to nothing) in the chain. In contrast, the gluteraldeldehyde coupling technique taught by Borel et al., Refers to causes of coupling at random locations along the chain and cross links. Thus, using that technique, chains greater than 20 base pairs can be coupled at an intermediate location in the chain to form chains dangling from substantially less than 20 base pairs in length, or the chains can be coupled together to form networks cross-linked of indefinite size.

Preferably, the polynucleotide duplexes of the conjugates of the invention are coupled or conjugated to the polymer at a location at or near one of its ends. Several strategic conjugations are available for linking the oligonucleotides to the biopolymer. The polynucleotide can be coupled to the polymer at the 3 'end of the polynucleotide, via a morpholine bridge, formed by the condensation of an oxidized 3' terminal ribose in one of the polynucleotide chains, with a free amino group in the polymer, and then subjecting the adduct to the condition reduction, to form the morpholine bonds. Such coupling requires that the polymer have at least an equal number of free amino groups (e.g., the DEK epsilon amino groups) for the number of polynucleotide duplexes to be linked to the polymer. The synthesis of such a conjugate was conducted in two raisins. The first step was the coupling of a chain of polynucleotide duplexes to the polymer, via the condensation / reduction reaction described above. The oxidized 3 '' terminal ribose is formed on the single polynucleotide chain by treating the chain with periodate to convert the 3 'terminal ribose group into an oxidized ribose group. The single-stranded polynuclephide is then added gently to an aqueous solution of the pH polymer of about · 6.0 to 8.0 at 2 - 8 ° C. The molar ratio of the polynuclefide to the polymer in all strategic conjugations will be usually in the range of about 2: 1 to about 30: 1, preferably about 5: 1 to 10: 1. During or after the condensation reaction (usually the reaction time from 24 to 48 hours), a strong reaction agent, such as sodium cyanoborohydride, was added to form the morpho group. The complementary strand of the female duplex is then added to the conjugate, and the female mixture is heated and gently cooled to give the strands to match. The conjugate can be purified by gel permeation chromatography.

Another strategy involves the formation of terminal aldehyde functional groups in the oligonucleotides, and using the functional groups to couple the oligonucleotide to the polymer, via the amino groups existing at that location. Advantages can be taken of the fact that the gem, the vertical diols, attached to the end

3 'of the oligonucleotide, can be oxidized with sodium periodate to produce aldehydes which can condense with the polymer's amino groups. When the diols are in a ring system, for example, a 5-membered ring, the resulting condensation product is a nitrogen-containing heterocyclic ring, for example, a six-membered morpholino or piperidine ring. The imino-condensation product was stabilized by reduction with a suitable reducing agent, for example, sodium borohydride or sodium cyanoborohydride. When the diol is cyclic, the resulting oxidation product contains only an aldehyde, and the condensation product is a secondary amine.

The vicinal diol strategy can also be continued by 5 * -terminal bonds. This is completed by the formation of cyanoethylphosphoramidite derivatives of a third hydroxy group in a triol, where the remaining hydroxy groups are vicinal, for example, 3,4-cis-dihydroxy, 1-hydroxymethylcyclo-pentane. In this specific case, the vicinal dihydroxy groups are blocked with dimethylSilane, and the primary dihydroxy group is achieved with 2ci anoethi 1 ™ N, N-dipropyl! 1 resulting chlorophosphoramide, Q derivative is used in the last step of synthesizing the standard oligonucleotide, and becomes residue 5 ^! '-terminal. After unblocking the oligonucleotide and removing the dimethyl group with the fluoride, acid, or base ion, the vicinal diol can be oxidized with periodate and condensed with amino groups, as above. A similar strategy can be followed for the acyclic triois to be used as 5 ^? -terminals.

Another procedure involves introducing alkylamino or alkyl sulfhydryl moieties, either within the 3 'or 5' ends of the oligonucleotide, by the appropriate nucleotide chemistry, for example, phosphoramidate chemistry. The nucleophilic groups can then be used to react with the large excess of homobifunctional cross-linking reagent, for example, dimethyl suberimide, in the case of alkyl amine derivatives, or an excess of heterobifunctional cross-linking reagent, for example, ester of m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), or succinimidyl (4-iodoacetyl) aminobenzoate (SXAB), for the derivatives here chi 1 sulphide. Once the excess crosslinker has been removed, the oligonucleotide derivatives will react with amine groups in the polymer.

Yet another strategy employs modified nucleosides. Suitable deoxynucleotide derivatives can be incorporated, by standard DNA synthetic chemistry, into a desired position in the oligonucleotide, preferably at the 5 'or 3' end. These nucleoside derivatives can then react specifically and directly with alkylamino groups in the polymer.

Alternatively, side reactions verified with the chemical of the chemical dialdehyde described above, such as amine-catalyzed beta-elimination, may be involved by the application of appropriate nucleoside derivatives, such as the 3 'terminus of the chain to be linked. An example of this is the 5'methylene extension of ribose; that is, a 5 'group (2-hydroxyethyl) instead of a group

5’hydroxymeti1. An alternative would be to use a phosphonate or phosphinate bond for the 3 'terminal dinucleotide of the oligonucleotide to be bound to the polymer.

The ability of the conjugates to act as a genetically tolerable SLE and to specifically suppress the production of anti-dsDNA antibodies can be assessed in the murine model described in the examples.

Conjugates will normally be formulated for administration by injection (for example, intraperitoneally, intramuscularly, etc.). Consequently, they will typically be combined with pharmaceutically acceptable aqueous carriers such as saline, Ringer's solution, dextrose solution, and the like. The conjugate will normally constitute about 0.01% to 107% by weight of the formulation. The conjugate is administered to an individual in sufficient quantities to at least partially restore tolerance to the SLE-causing antigens. Such amounts are sometimes referred to here as therapeutically effective amounts. The particular dosage regimen, that is, the dose, timing and repetition, will depend on the individual in particular, and his individual medical history. Normally, a dose of about 1 to 1000 pg of conjugate / kg of body weight will be given. Repetitive administrations may be required to achieve and / or maintain an immune tolerance state »

The following examples further illustrate the invention and its unforeseen characteristics relating to the prior industry. These examples are not intended to limit the invention in any way.

Ρ - ΈΚ Conjugate and Nucleoside Test

Individuals

As previously indicated, the development of the inventive conjugates was preceded by tests that showed that the conjugates of D-EK and individual nucleotides did not tolerate the anti-DNA response in the murine model for SLE (mice of the species (NZB! ·: NZW ) F).

A lot of D-EK were obtained from

BioMakor / Yeda (Rehovet, Israel), Their relative weights were standardized against known proteins by gel permeation chromatography. Material was desalted and measured by dialysis 25 Kd cutoff tubes against 0, IM K HPO, pH 9.5 molecular globular HPLC , and exhaustive in, the material was then dialyzed twice against water. This material is stored in buffer with 0, IM of K HF'0, pH 9.5, at 4 ° C. The average molecular weight of the products was determined by physical methods, including sedimentation balance, F'AGE, and HPLC-GPC, and low-angle diffusion, and it was found to be approximately 28,000, Amino acids analyzed by acid hydrolysis showed that the copolymer had 607 glutamic acid, and 407 «lysine.

The conjugates of D-EK and riboadenosine, riboguanosine, ribocytosine and ribotimidine were essentially prepared as described in Eshar et al, J. Imm. (1975) 114: 872. A mixture of equal parts of each of these conjugates (called nucleosides D-EK) was used in the following tests.

Two groups of 6 17 week old (NZB x NZW) FIM female rats were injected i.p., either with a saline purgative or 1 mg / rat D-EK nucleoside per day for three days. Seven days later the mice were bleeding. Two weeks later, the treatment was repeated. Seven days later the «SSís» rats were bleeding. The sera from the first and second bleeds were tested for anti ssDNA antibodies using the following antigen-specific ELISA protocol immobilized in polystyrene dish containers and reacts with antibodies in the rat lprus sera (dpr / lpr). Anti-ssDIMA antibodies are visualized by adding antibodies bound by specific enzymes to the immunoglobulin isotypes bound to ssDNA in the dish. Subsequent addition of the enzyme substrate results in a color reaction, which is performed on a spectrophotometer.

ssDNA fe is prepared from dsDIMA of calf thymus. The commercially obtained veal thymus dsDNA was treated with S-1 nuclease to obtain homogeneous dsDIMA. The dsDNA is boiled for 5 min. in a water bath, and cooled quickly in an ice bath. Each ssDNA cooking was prepared immediately before use in the experiment.

Ninety-six flat-bottomed dishes in the container are exposed to ultraviolet (UV) light overnight in a Steril Gard Hood before use. The dishes in the dishes are coated the day before at 4 ° C with 100 µl of ssDNA, at a concentration of 1 pg / ml, in a salt containing 10 pg / ml of bovine serum methyl albumin. The next morning, the dishes are washed once, in dissolved saline phosphate (PBS), and are then blocked by placing 200 pl of 17% bovine serum albumin in PBS (PBSA) in each container, for 45 min »at 37 ° C. After blocking, the dishes are washed a second time in PBS and dried quickly. Then, 100 µl sera test and contract dilutions of sera, diluted in PBSA at 17. and containing 0.57 µm. of Tween-20, are placed

1?

In suitable containers. The plates are incubated for 1 hour at 37 ° C. Then they are washed 5 times in PBS and dried rapidly, followed by the addition of one hundred microliters of alkaline phosphatase antibody to anti-rat conjugate UgG, A and M). The dishes are incubated for another hour at 37 ° C. The dishes are washed 7 times in PBS and dried quickly. Then, 50 µl of 1-x enzyme substrate is added, and the dishes are incubated at room temperature for one hour. The reaction ends with the addition of 50 µl of disodium hydrogen phosphate, pH 8.6. The optical density value at 550 nm is determined for each container with a Titertek spectrophotometer »

The data is shown in Figure 1. As shown, the nucleoside D-EK has no detectable effect on anti-ssDNA titers in mice.

Example 2

F'olinucleotide test for Antisera SLE binding activity.

In addition to the polynucleotides used in the conjugates of the invention, several other DNAs were prepared and tested for their binding activity with SLE antisera. These tests, described below, show that the reactivity of the polynucleotides of the inventive conjugates was unpredictable and unexpected.

Various single- and double-stranded polynucleotides have been prepared by chemical synthesis and, where appropriate, enzymatic extension and / or stemming. Chemical oligonucleotide synthesis was performed on a Pharmacia Gene Assembler, using an adjustable Cruachem column, and triester phosphite chemistry. The solid phase was porous controlled crystals of 500 angstroms which were derived with the appropriate 3'-riba or 3 ^: '-deoxy nucleotide. The oligonucleotides were purified by simple dialysis »In the case of oligonucleotides longer than '70 bases, individual chains were phosphorylated using ATP and poly nucleotide chi nase rT4» After desalination on a Pharmacia PD10 column, the bound phosphoryl chains were covalently coupled using rT4 DNA ligase. All strands share a common 5 'CATO end sequence, which provides a single viscous end »Where appropriate, single strands have been paired to form dsDNA.

Two assays were employed to determine the binding of polynucleotides with antiserum lupus (1) a modified Farr assay, in which the radiolabeled DNA is precipitated from the solution after being bound to the antibody, and (2) an ELISA. In a mold, 25 µl of each antiserum dilution was previously prepared in a dissolved Tris salt (TBS, 0.151 * 1 NaCl, 0.011 * 1 Tris, pH 7.5) containing 0.1 mg / ml human gamma globulin. Stations were diluted with 125

125 pl TBS, 50 pl Ι-dsDNA (Diagnostics Products Corp., Los

Angeles, CA) were added to each sample, and the samples were incubated at 37 ° C for ^{! <} Z hour »Then, 500 µl of saturated (NH) SC) was added, the sample was incubated at 4 ° 4 2 4

C for 15 min »and centrifuged» The radioactivity of the supernatant was measured in a gamma counter. The depletion of the supernatant's radioactivity was a direct measure of the antibody concentration in the solution. In the ELISA, the dish containers were coated at 4 ° C with 100 µl of 10 µg / ml dsDNA in saline, containing 10 µg / ml of methylated BSA. The containers were washed with PBS and then blocked by placing 200 µl of 17% BSA in 1'BS (PBSA) in each container for 45 min at 37 ° 0. The dishes were again washed with PBS. Then, 100 µl of test sera diluted in PBSA at 17 ° C, containing 0.57 Tween 20, was added. For inhibition studies, the inhibitor (for example, a polynucleotide) was also added. The dishes were then incubated for 1 hour at 37 ° C and washed with PBS. Goat antibody labeled with alkaline phosphatase, 100 µl / cell, was added and the dishes incubated for another hour at -37 ° C. The dishes were then washed, the substrate was added, and the dishes were incubated in an oven for one hour. The reaction was stopped by the addition of disodium hydrogen phosphate and the dishes were read with a spectrophotometer.

Tables 1 and 2 below show, respectively, several single-stranded polynucleotides and double-stranded polynucleotides that do not significantly inhibit dsDNA, linked by SLE autoantibodies. in these tests.

TABLE 1

SIMPLE CHAIN NUCLEPTIDIC HOMOPOLYMERS____ BELOW

500nM DOES NOT SIGNIFICANTLY INHIBIT MURINE-CONNECTED dsDNA (MRL) QU HUMAN SLE AUTO-ANTIBODIES n-mer composition n-mer composition

A. Homopurines poly d (G) n

1219 * 350 *

Q poly d (A) n

390 * 60 OX 22 12 i

B. Homopyrimidines poly d (C) n

32? * poly d (T) n

Χ.Χ.7

* Enzymatically synthesized, using rT4 DNA polymerase. Because molecular weights are a distribution, the values of n for enzymatically synthesized oligomeres constitute an average weight number, estimated from the Sw value, 20 of each.

TABLE 2

EXAMPLES OF DUPLEXES 0LIG0NUCLEQTIDQ5 UP TO 32 PAIRS OF BASES THAT

BELOW 500 nM DOES NOT SIGNIFICANTLY INHIBIT THE CONNECTION OF THE dsDNA TO MURINE (MRL) OR HUMAN ANTISERA

A, HOMOPOLYMERS

Regular

Examples: EA3: ET3, EG3: EC3, EI3: EC3

30 25 25 20 20

B. HETEROPOLIMEROS

1. Self-alignment

Example: EG3 -EA3 -EC3; EG3 -ET3 -EC3 2 10 2 2 10 2

2 · Repeating plmeros

Examples: EAT3: EAT3, EAC3: EGT3

16 10 10

3. Repeating trimers

Examples; ETTC3: EGAA3, ETTG3: ECAA3 8 8 8

4. Repeating tetralmers! Example: EACGT3: EACGT3

TABLE 3

EXAMPLES OF DUPLEXES 0L.160NUCLEDTIDDS THAT UNDER 500 nM <I less than 500 nM) INHIBIT SIGNI FL'CAT 1'VAMINALLY THE CONNECTION OF THE dsDNA

AQ HUMAN SERUM SLE AND AP MURINA SERUM (MRL)

Composition n Greater than Length  of the Oligomer - - - - -------------- d (AC) : d (TG) 20 40 or bigger n n d (AT) : d (TA) 20 40 or bigger n n d (CI) : d (CI) 20 40 or bigger n n d (AC) : d (TG) 20 40 or bigger n n d (AG) : d (TC) 20 40 or bigger n n d (ATC) : d (GAT) 15 45 or bigger n n d (TAC) sd (GTA) 15 45 or bigger n n Example 3

Correlation of Liaison Activity with the CD Spectrum

The spectral measurements of the CD were carried out in poly (AT): poly (AT) of about 228 bp in length (for example from A-DNA), poly (GC): poly (GC) of approximately 330 bp in length (for example, Z-DNA), salmon sperm DNA with an average length greater than 1200 bp (an example of native DNA with a B-DNA type with helical configuration) and the duplex (AC): (TG) described above. DLE antisera SLE 30 30 binding assays on these oligonucleotides and DNA were carried out using the modified Parr assay as above.

All DNA's and oligonucleotides were dissolved in standard solvent (0.15M NaCl, 0, IM sodium citrate, pH 7.0), and their relative abilities to bind to the autoimmune sera H-SLE were compared to theirs. respective capabilities to absorb circularly polarized monochromatic light to the left and right (CD spectroscopy). Serological data are expressed as the ability to inhibit the binding of dsDNA to sera; the spectrum is presented as a molar ellipticity by nucleotide residue:

COZI = 100 / c.L.

where 9 is the ellipticity observed in degrees, L is the length of the cell routing in cm, and c is the concentration in moles of nucleotides per liter.

Figure 2 shows the CD spectrum of poly (AT): poly (AT).

'Figure 3 shows the CD spectrum of poly (GC): poly (GC) (solid line interrupted with solid circles), salmon sperm DNA (dashed line), and duplex (AC): (TG) ( line

30 is still full).

Figure 4 shows the relative capacities of the different forms of DNA to bind to the SLE antisera. As shown, synthetic type B DNA has similar reactivity to type B DNA (the last calf DNA was used), and substantially greater reactivity than any type A or type Z DNA. (The helical type was characterized by the CD spectrum and , as indicated above, may not be conclusive.)

Synthesis of the D-EK Conjugate of (AC); (TG) .30 30

Based on the binding activity and stability, the duplex (AC): (TG) described above was selected for

30 genetic tolerance studies. A conjugate of this duplex and a D-EK copolymer was prepared using the preferred synthetic procedure, outlined above. The details of this synthesis follow.

^ 5 rj »« «25saae2 ^{, a} ' ^{sS! w} '. * ^eai

-j

60:40,

MW med copolymer D-EK, G: L mol 30,000 daltons was prepared with a ratio of starting from the material obtained from BioMakor, Rehovet, Israel. This copolymer was dialyzed against 0.1 M KHCO, pH 9.5, in dialysis tubes with a degree of

T admittance of molecular weight of 25,000 daltons, for a final concentration of 20 mg / ml, as determined by the absorbance at 220 nm in a 1 cm cuvette, where:

A (30,000 mg / nmol) (168,000 mL / cm nmol) □ (AC) was synthesized on a DNA synthesizer and dialyzed against deionized water in dialysis tubes with a molecular weight intake grade of 12,000 - 14,000 daltons »The resulting solution is adjusted to a final concentration of 35 mg / ml, as determined by absorbance at 260 nm in an i cm cuvette, where

A (18.106 mg / nmol)

260 <flC> ^ mg / ml (458,160 ml / cm nmol)

A 0.1M aqueous solution of sodium periodate and water was added to the (AC) to give a mixture reaction with a 5: 1 molar excess of the periodate for DNA. The mixture was stirred well and then placed at 4 ° C for 15 min. The excess periodate was then precipitated by the addition of an excess of potassium chloride and the precipitate was removed by centrifugation.

A solution of D-EK and sodium cyanoborohydride was pipetted into a polypropylene reaction vessel and the pH was adjusted between 6.0 and 8.0. The oxidized (AC) was added dropwise to 30

D-ER in a weight ratio of 6.035: 1 (molar ratio of conjugation o

qs of IOsl), with vigorous stirring for 24 - 48 hours at 4 ° C. After condensation, solid sodium borohydride was added to the reaction mixture, with stirring, until a final concentration of 1.0 mg / ml was reached. The reaction vessel was more or less encapsulated and allowed to remain, without being stirred, for at least 30 min. The reaction mixture was then transferred to the dialysis tubes with an intake degree of 50,000 daltons and was dialyzed extensively against 0.21 * 1 sodium citrate, pH 5.0, at 4 ° C.

The conjugate was then purified on a Sephacryl S-200 gel permeation chromatographic column, in 0.21 * 1 sodium phosphate, 0.51 * 1 sodium chloride, pH 7.2. Fractions were analyzed for oligonucleotide concentration by

0D, and the concentration of D-EK by a trinitrobenzene sulfunate 260 assay (Albers, R. W., et al., Analyt Biochem (1983) 137s437-443). The separation of the '-'X-free oligonucleotide conjugate was determined by a 5'hydroxyl labeled P-kinase by the oligonucleotide chain, followed by a polyacrylamide a

107., 81 * 1 of urea sequencing gel, and autoradiography. The gel was cut and counted in a liquid scintillation beta counter, and those fractions exhibiting i 957. purity were pooled and dialyzed against 0.011 * 1 sodium citrate, 0.151 * 1 sodium chloride, pH 7.0 (solvent of formulation) in preparation for staging.

(TG) was prepared as, described above and dialyzed against the formulation solvent, in the same way as (AC). The molar nucleotide concentration (l * INC) of (TG)

30 was determined by measuring the absorbance at 260 nm in a 1 cm cuvette * INC (TG)

A / (9164 mL / cm mmol)

260 nm

lnNC of the (AC) -D-EK conjugate was determined by measuring the absorbance of the dialyzed solution at 260 nm ·.

MNC (AC) -D-EK = A / (7636 mL / cm mmol)

260 nm (TG) was then paired with the 30 (AC) -D-EK conjugate as follows »An equal (TG) MNC was added 30 30 to the (AC) -D-EK limiting reagent in a 30 propylene or glass. The mixture was heated to> 95 ° C in a water bath, which was maintained between 95 ° C and 98 ° C for 10 min. » The solution was then gently cooled to room temperature at a rate of 10 ° / hour.

The paired product is dialyzed against the formulation solvent in dialysis tubes with a molecular weight inlet grade of 50,000 daltons. After extensive dialysis, the final conjugate is sterile filtered through a 0.22 µm membrane. It is characterized by UV spectroscopy, liquid chromatography with high-resolution permeation gel, electrophoresis with polyacrylamide gel and thermography before sterile filling.

Example 5

(TG) 50 Conjugate Testing; (AC) -D-EK as a 50 50

Tolerance

(TG) s (AC) -D-EK conjugate, described above, was

30 tested in the murine iRL model (lpr / lpr) for human SLE. A genetic defect in this species of rat leads to hyperproliferation of T helper cells which, in all possibilities, participate in autoreactive differentiation of B cells. These results in secretion of antibodies to DNA, as well as a plethora of other antibodies. As previously indicated, antibodies to dsDNA are a contrast to human SLE and their presence correlates well with disease severity and renal pathology in humans.

conjugate was diluted in saline to the desired concentration, for i.p. in rats. Four groups of five rats with 12 to 14 weeks of age each were used. The rats bled on the morning of day 1 and were injected in the afternoon. Consequently, the mice bled in the morning and were injected in the afternoon each week for a further five weeks. In weeks 6 and 7 the mice only bled. Group 1 (control) was injected with 0.3 mg of D-EK copolymer / week / rat; Group 2, with 0.1 mg of the conjugate / week / rat; Group 3, Group 4, with 0.3 mg of the conjugate / week / rat; and conjugate / week / rat.

The plasma samples taken from the rats were diluted 1:10 and 1:50 in Tris solvent (0.11 * 1, pH 7.4) and used with

1.0 mg of in the modified Farr assay described above, using H-dsDNA in

125 instead of Ι-dsDNA to determine the titer of the antidsDNA antibody in the samples. The data obtained in the modified Farr assay have been converted to antigen binding capacity and are plotted in Figure 5 (the conjugate is called LJP-105).

Four weeks after the end of treatment, two mice from each group and one mouse from the control group were sacrificed, and secretion levels of anti-dsDNA antibodies in each group were determined in a spleen cell by ELISA where

4 x 10 to 1.5 x 10 spleen cells in double dilutions were placed in each cell. The results of these tests are reported in Figure 6.

A conjugate experiment was also performed on older MRL rats, 22 to 24 weeks old. Again, the rats were dosed by i.p. once a week for four weeks. The serological levels of anti-dsDNA were determined after one month of treatment, and were compared to the values before bleeding obtained at the beginning. These data, expressed as a percentage change in antigen-binding capacity (ABC) for individual mice, are shown in Figure 7. Figure 7a shows the average data for these tests. The variability in rats by dosage group (0.01, 0.1, 0.3 and 1.0 mg / rat conjugates; control rats received a mixture of carrier polymer and substituted unconjugated nucleic acid) reflects deaths during the experience.

The spleen cell test data from the therapeutic experiment are shown in Figure 8, again demonstrating a significant difference between control and conjugate-treated rats, and confirm previous serological results. As a control experiment, soluble dsDNA was shown to inhibit spleen cell assay. In addition, adding the spleen cells of polynucleotide-treated animals to the spleen control cells does not decrease in color; preferably, the effect was additive, thus determining that that boundary cell conjugate inhibited the assay.

Finally, it was shown that the conjugate can be effective via ip, im, or iv Female MRL rats, twenty-two weeks old, were dosed with 0.1 mg of conjugate per week for four weeks, and the percentage change was measured in the antigenic binding capacity for anti-dsDNA. While the control mice did not increase as much as had been seen with other experiments, there were significantly higher anti-dsDNA titers in the mice dosed subcutaneously, on the one hand, and the mice dosed with the other conjugated via ip, im, or iv, for another.

Example 6

This example illustrates the alternative procedures for making the conjugate (AC): (ΤΘ) -D-EK.

30 i

Closing of 60 mers

Molecular cloning is used to make 60 mers according to the following protocol. A 64-mer, consisting of the sequence

S ^ AATTC (ST) S3 · and a second 64-mer sequence 5'TCGAC 29 (AC) G3 'are synthesized and phosphorylated by standard methods. The 29 oligomers are mixed in equal molar ratios, and cooled gently to allow double formation and the occurrence of oligomerization. The protrusions of the oligomers are paired due to the superposition of 4 bases of the first oligomer, creating an EcoRI location, and the second protrusion creating a location position Sal I (the same as the HincII location). After slow cooling, the mixture is linked by standard methods to form an oligomer of 60 mer linked units, covalently separated, either by an EcoRI position or by a Sal. The oligomeric mixture is ligated into a pUC19, which has been previously digested with EcoRI and Sal. The ligation mixture is introduced into E. Coli <JM107 by transformation.

Resistant ampicillin colonies are collected, developed and isolated from the plasmid DNA. The insertion size is determined with restricted digestion. The desired clone has an insert comprising at least half of the plasmid or> 50, 60 mer units.

The resulting clone is developed on a large scale and isolated from the plasmid. The plasmid is digested with EcoRI and Hindi to release 60 mer with a 4 base EcoRI overhang and a sharp end at the opposite end and generated by Hindi. The oligomers are purified and paired with D-EK which has the 4 base oligomer 3'TTAA-P with a 5 'phosphate covalently linked to D-EK through 3 ^; 'T. The 60 mers are paired and covalently linked to D-EK / TTAA by ligase.

PCR production of 60 mer

The polymerase chain reaction is used to synthesize 60 mers to couple to D-EK, by the methods described in the references cited above.

In summary, (GT) is chemically synthesized with 30 small random sequences such as GACT and CTGA, at the extreme

5 ^! 'and 3' of (GT), respectively (as illustrated below). The 30 small random strings are long enough to ensure characteristic primer records for the model. The primer contains the sequence of the random sequence, plus several extra repetitions of the GT as needed for stability in the emparahagem reaction. One of the primers ”also has an extra base modified at the 5 'end, which allows chemical coupling to the DEK.

The PCR reaction is carried out for at least 20 cycles according to the methods mentioned above. The oligomers produced by PCR are purified by chromatography, just like the

HPLC, and then conjugated to D-EK by one of the procedures described above.

PRIMER ls (CA) -GACTS '

MODEL 1: 5’fcNGACT- (ST) -CTGA3 ’

PRIMER 2: 5 ¹¹ * GACT- <GT)

MODEL 2: 3 ^? CTGA- (CA) -TACGõ '= modified base for coupling to D-EK

Claims (14)

CLAIMS ί- A conjugate of (a) a biologically stable polymer, and (b) a multiplicity of polynucleotide duplexes of at least 20 base pairs, each linked to the polymer, characterized by the fact that said duplexes each have bound, a significant binding activity for human systemic lupus erythematosus double-stranded autoantibodies. 2- A conjugate as claimed in claim 1, characterized in that the biologically stable polymer is a copolymer of D-glutamic acid (E) and D-lysine (K) having a molecular weight of about 5,000 to about 50,000 and an E: K mole ratio of approximately 60:40. 3- ·· A conjugate, as claimed in King VI ndication 1 or 2, characterized by the fact that the duplexes are substantially homogeneous in length. A conjugate as claimed in claim 3, characterized in that the duplexes are substantially homogeneous in the nucleotide composition. 5- A conjugate as claimed in claims 1, 2, 3 or 4, characterized in that the duplexes are 30 to 250 bp in length. 6- A conjugate, as claimed in king vindications 1, 2, 3, 4 or 5, characterized in that the duplexes are connected to the polymer at, near one of its ends. A conjugate as claimed in claim 6, characterized in that the duplexes are attached to the polymer through a reaction between a functional group at, or near the terminus of a duplex chain and the free amino groups in the polymer. 8- A conjugate, as claimed in claims i, 2, 3, 4, 5, 6 or 7, characterized in that the polynucleotide duplexes are composed of complementary mer repeating units, from 2 to 4 different bases. A conjugate as claimed in claim 8, characterized in that the polynucleotide duplexes are: poly d (GC)! poly d (CB>; poly d (AT>: poly d (TA>; poly d (IC): poly d (CP); poly d (AC) spoli d (TG); or poly d (AS) spoli d (TC). A conjugate as claimed in claim 1 or 2, characterized in that the polynucleotide duplex is (AC); (TS). 30 30 A pharmaceutical composition for treating lupus, comprising the conjugate of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, characterized in that it is formulated with an injectable, pharmaceutically acceptable carrier. 12- A single-stranded polynucleotide of at least 20 bases, having a functional group at or near one of its terminus that will react with a free amino group, when paired with a complementary single-stranded polynucleotide, characterized by the fact that have significant binding activity for human systemic lupus double-stranded anti-DNA autoantibodies. 13. A single-stranded polynucleotide as claimed in claim 12, characterized in that it consists of a repeating mer unit, from 2 to 4 different bases. 14. A method for obtaining the conguguado of claim 1, characterized in that it comprises: (a) the reaction of a multiplicity of single-stranded polynucleotides of at least 20 base pairs, each of which has an amino-reactive functional group at or near its terminus, forming the free amino groups, in the polymer , a conjugate, (b) the pairing of complementary single-stranded polynucleotides with the polymer-conjugated single-stranded polynucleotides to form double-stranded pairs.